Silencing of HOXC9 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs).
(A) Overall morphology of 48 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B) and (C). (B) Normal formation of the PLs (arrows) in 48 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C) Silencing of HOXC9 expression using 2 ng translational-blocking morpholino disrupted the formation of the PLs (asterisks) in 48 hpf tg(fli1:EGFP) zebrafish embryo. (D) Quantification of 48 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. (E) RT-PCR analysis for increased expression of Stab2 in zebrafish driven by mRNA (50 pg) mediated overexpression of HOXC9. (F) Quantification of (E), n = 3 per group. *p<0.05 vs.Orange-mRNA. (G) RT-PCR analysis for reduced expression of Stab2 in zebrafish driven by morpholino (2 ng) mediated silencing of HOXC9. (H) Quantification of (G), n = 4 per group. *p<0.05 vs.Co-Mo. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of Stab2 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs).
(A) Overall morphology of 48 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 48 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C,D) Silencing of Stab2 expression using 4 ng splice-blocking morpholino targeting exon 2 (C) or 2 ng splice-blocking morpholino targeting exon 9 (D) disrupted the formation of the PLs (asterisks) in 48 hpf tg(fli1:EGFP) zebrafish embryos. (E) Quantification of 48 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. (F) Functionality of the splice blocking morpholino Stab2-Ex2-Mo. RT-PCR of control morpholino (4 ng) and Stab2-Ex2-Mo (4 ng) injected zebrafish embryos at 24 hpf. (G) Functionality of the splice blocking morpholino Stab2-Ex9-Mo. RT-PCR of control morpholino (4 ng) and Stab2-Ex9-Mo (2 ng) injected zebrafish embryos at 24 hpf. WT: wild type, morph: morphant. Black scale bar: 500 μm. White scale bar: 50 μm.

HOXC9 overexpression rescues the defects in parachordal lymphangioplast (PL) formation in Stab2 morphants.
(A) Overall morphology of 48 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 48 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C) Silencing of Stab2 expression using 4 ng splice-blocking morpholino disrupted the formation of the PLs (asterisks) in 48 hpf tg(fli1:EGFP) zebrafish embryo. (D) Injection of HOXC9 mRNA (50 pg) rescued the Stab2 loss-of-function phenotype in 48 hpf tg(fli1:EGFP) zebrafish embryo. (E) Quantification of 48 hpf tg(fli1:EGFP) fish embryos showing a disturbed PL formation including rescue experiments using HOXC9 mRNA (50 pg). Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. (F) Functionality of the mRNA injection. Western Blot of Orange-mRNA (50 pg) and HOXC9-mRNA (50 pg) injected zebrafish embryos at 24 hpf. (G) Quantification of (F), n = 3 per group. *p<0.05 vs. Orange-mRNA. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of Stab1 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs).
(A) Overall morphology of 48 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B-D). (B) Normal formation of the PLs (arrows) in 48 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C,D) Silencing of Stab1 expression using 4 ng splice-blocking morpholino targeting exon 3 (C) or 12 ng splice-blocking morpholino targeting exon 4 (D) disrupted the formation of the PLs (asterisks) in 48 hpf tg(fli1:EGFP) fish embryos. (E) Quantification of 48 hpf tg(fli1:EGFP) fish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. (F) Functionality of the splice blocking morpholinos Stab1-Ex3-Mo and Stab1-Ex4-Mo. RT-PCR of control morpholino (4 ng), Stab1-Ex3-Mo (4 ng) and Stab1-Ex4-Mo (12 ng) injected zebrafish embryos at 24 hpf. WT: wild type, morph: morphant. Black scale bar: 500 μm. White scale bar: 50 μm.

HOXC9 overexpression rescues the defects in parachordal lymphangioplast (PL) formation in Stab1 morphants.
(A) Overall morphology of 48 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 48 hpf tg(fli1:EGFP) fish embryo after injection of 4 ng control morpholino. (C) Silencing of Stab1 expression using 4 ng splice-blocking morpholino disrupted the formation of the PLs (asterisks) in 48 hpf tg(fli1:EGFP) zebrafish embryo. (D) Injection of HOXC9 mRNA (50 pg) rescued the Stab1 loss-of-function phenotype in 48 hpf tg(fli1:EGFP) zebrafish embryo. (E) Quantification of 48 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation including rescue experiments using HOXC9 mRNA (50 pg). Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. (F) RT-PCR analysis for increased expression of Stab2 in zebrafish injected with the Stab1-Ex3-Mo (4 ng) and HOXC9 mRNA (50 pg). (G) Quantification of (F), n = 3 per group. *p<0.05 vs. Co-Mo. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of HOXC9, Stab1 and Stab2 expression in zebrafish inhibits formation of the thoracic duct (TD).
(A) 120 hpf tg(fli1:EGFP) zebrafish embryo. Blue box marks the region magnified in (A2). (A2) Trunk vasculature of the embryo shown in (A). Red box marks the region magnified in (B–J). (B) Normal formation of the TD (arrows and dotted line) in 120 hpf tg(fli1:EGFP) zebrafish embryos after injection of control morpholino. Blue bar marks dorsal aorta and red bar marks cardinal vein. (C–G) Silencing of HOXC9, Stab1 and Stab2 expression using the indicated morpholinos disrupted the formation of the TD (asterisks) in 120 hpf tg(fli1:EGFP) zebrafish embryos. (H–J) Co-injection of 50 pg HOXC9 mRNA rescued the defects in TD formation caused by silencing of HOXC9, Stab1 and Stab2 using the indicated morpholinos. (K) Quantification of defects in TD formation of embryos shown in (A–J) including rescue experiments with 50 pg HOXC9-mRNA. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of HOXC9, Stab2 and Stab1 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs).
(A–F′) Whole mount antibody staining against GFP in 48 hpf (A–F) and 72 h hpf (A′–F′) tg(fli1:EGFP) zebrafish embryos injected with the indicated morpholinos. (A,A′) Normal formation of the PLs (arrows) in 48 hpf (A) and 72 hpf (A′) tg(fli1:EGFP) zebrafish embryos after injection of 4 ng control morpholino. (B–F′) Silencing of HOXC9, Stab2 and Stab1 expression using the indicated morpholinos disrupted formation of the PLs (asterisks) in 48 hpf (B–F) and 72 hpf (B′–F′) tg(fli1:EGFP) zebrafish embryos. Black scale bar: 100 μm.

Silencing of HOXC9 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs) at 72 hpf.
(A) Overall morphology of 72 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B) and (C). (B) Normal formation of the PLs (arrows) in 72 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C) Silencing of HOXC9 expression using 2 ng translational-blocking morpholino disrupted the formation of the PLs (asterisks) in 72 hpf tg(fli1:EGFP) zebrafish embryo. (D) Quantification of 72 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of Stab2 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs) at 72 hpf. (A) Overall morphology of 72 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 72 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C,D) Silencing of Stab2 expression using 4 ng splice-blocking morpholino targeting exon 2 (C) or 2 ng splice-blocking morpholino targeting exon 9 (D) disrupted the formation of the PLs (asterisks) in 72 hpf tg(fli1:EGFP) zebrafish embryos. (E) Quantification of 72 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of Stab2 expression using an ATG-morpholino in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs). (A;D) Overall morphology of 48 hpf (A) and 72 hpf (D) zebrafish embryos after injection of control morpholino. Red box shows region displayed below in (B,C,E,F). (B,E) Normal formation of the PLs (arrows) in 48 hpf (B) and 72 hpf (E) tg(fli1:EGFP) zebrafish embryos after injection of 4 ng control morpholino. (C,F) Silencing of Stab2 expression using 2 ng translational-blocking morpholino disrupted the formation of the PLs (asterisks) in 48 hpf (C) and 72 hpf (F) tg(fli1:EGFP) zebrafish embryos. (G,H) Quantification of 48 hpf (G) and 72 hpf (H) tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

HOXC9 overexpression rescues defects in parachordal lymphangioplast (PL) formation in Stab2 and Stab1 morphants.
(A–E′) Whole mount antibody staining against GFP in 48 hpf (A–E) and 72 hpf (A′–E′) tg(fli1:EGFP) zebrafish embryos injected with the indicated morpholinos. (A,A2) Normal formation of the PLs (arrows) in 48 hpf (A) and 72 hpf (A′) tg(fli1:EGFP) zebrafish embryos after injection of 4 ng control morpholino. (B,B′) Silencing of Stab2 expression using 4 ng Stab2-Ex2-Mo disrupted formation of the PLs (asterisks) in 48 hpf (B) and 72 hpf (B′) tg(fli1:EGFP) zebrafish embryos. (C,C′) Injection of HOXC9 mRNA (50 pg) rescued the Stab2 loss-of-function phenotype in 48 hpf (C) and 72 hpf (C′) tg(fli1:EGFP) zebrafish embryos. (D,D′) Silencing of Stab1 expression using 4 ng Stab1-Ex3-Mo disrupted formation of the PLs (asterisks) in 48 hpf (D) and 72 hpf (D′) tg(fli1:EGFP) zebrafish embryos. (E,E′) Injection of HOXC9 mRNA (50 pg) rescued the Stab1 loss-of-function phenotype in 48 hpf (E) and 72 hpf (E′) tg(fli1:EGFP) zebrafish embryos. Black scale bar: 500 μm.

HOXC9 overexpression rescues the defects in parachordal lymphangioplast (PL) formation in Stab2 morphants at 72 hpf.
(A) Overall morphology of 72 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 72 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C) Silencing of Stab2 expression using 4 ng splice-blocking morpholino disrupted the formation of the PLs (asterisks) in 72 hpf tg(fli1:EGFP) zebrafish embryo. (D) Injection of HOXC9 mRNA (50 pg) rescued the Stab2 loss-of-function phenotype in 72 hpf tg(fli1:EGFP) zebrafish embryo. (E) Quantification of 72 hpf tg(fli1:EGFP) fish embryos showing a disturbed PL formation including rescue experiments using HOXC9 mRNA (50 pg). Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

Low dose injection (50 pg) of HOXC9 mRNA shows no effect on zebrafish vascular morphology.
(A,D) Overall morphology of 48 hpf (A) and 72 hpf (D) zebrafish embryos after injection of control morpholino. Red box shows region displayed in (B,C,E,F). (B,E) Normal formation of the PLs (arrows) in 48 hpf (B) and 72 hpf (E) tg(fli1:EGFP) fish embryos after injection of 4 ng control morpholino or 4 ng control morpholino combined with 50 pg HOXC9 mRNA (C,F). (G) RT-PCR analysis of zebrafish lysates injected with indicated morpholinos showed no regulation of stabilin 1 by HOXC9 silencing. (H) RT-PCR analysis of zebrafish lysates injected with indicated mRNA showed no regulation of stabilin 1 by HOXC9 overexpression. Black scale bar: 500 μm. White scale bar: 50 μm.

Silencing of Stab1 expression in zebrafish inhibits assembly of parachordal lymphangioplasts (PLs) at 72 hpf.
(A) Overall morphology of 72 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 72 hpf tg(fli1:EGFP) zebrafish embryo after injection of 4 ng control morpholino. (C,D) Silencing of Stab1 expression using 4 ng splice-blocking morpholino targeting exon 3 (C) or 12 ng splice-blocking morpholino targeting exon 4 (D) disrupted the formation of the PLs (asterisks) in 72 hpf tg(fli1:EGFP) fish embryos. (E) Quantification of 72 hpf tg(fli1:EGFP) fish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

HOXC9 overexpression rescues the defects in parachordal lymphangioplast (PL) formation in Stab1 morphants. (A) Overall morphology of 72 hpf zebrafish embryo after control morpholino injection. Red box shows region displayed in (B–D). (B) Normal formation of the PLs (arrows) in 72 hpf tg(fli1:EGFP) fish embryo after injection of 4 ng control morpholino. (C) Silencing of Stab1 expression using 4 ng splice-blocking morpholino disrupted the formation of the PLs (asterisks) in 72 hpf tg(fli1:EGFP) zebrafish embryo. (D) Injection of HOXC9 mRNA (50 pg) rescued the Stab1 loss-of-function phenotype in 72 hpf tg(fli1:EGFP) zebrafish embryo. (E) Quantification of 72 hpf tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation including rescue experiments using HOXC9 mRNA (50 pg). Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.

Expression silencing of Stab1 and Stab2 shows no additive effect on parachordal lymphangioplast (PL) assembly.
(A,D) Overall morphology of 48 hpf (A) and 72 hpf (D) zebrafish embryos after injection of control morpholino. Red box shows region displayed in (B,C,E,F). (B,E) Normal formation of the PLs (arrows) in 48 hpf (B) and 72 hpf (E) tg(fli1:EGFP) zebrafish embryos after injection of 4 ng control morpholino. (C,F) Double silencing of Stab1 and Stab2 expression using 4 ng of each splice-blocking morpholino for Stab1 and Stab2, respectively, disrupted formation of the PLs (asterisks) in 48 hpf (C) and 72 hpf (F) tg(fli1:EGFP) zebrafish embryos. (G,H) Quantification of 48 hpf (G) and 72 hpf (H) tg(fli1:EGFP) zebrafish embryos showing a disturbed PL formation. Embryos were divided in three groups depending on the PL appearance being completely absent, partially formed or completely present. Black scale bar: 500 μm. White scale bar: 50 μm.